Low vitamin C and increased oxidative stress and cell death in mice that lack the sodium-dependent vitamin C transporter SVCT2.

The sodium-dependent vitamin C transporter (SVCT2) is responsible for the transport of vitamin C into cells in multiple organs, from either the blood or the cerebrospinal fluid. Mice null for SVCT2 (SVCT2(-/-)) do not survive past birth but the cause of death has not yet been ascertained. After mating of SVCT2(+/-) males and SVCT2(+/-) females, fewer SVCT2(-/-) and SVCT2(+/-) progeny were observed than would be expected according to Mendelian ratios. Vitamin C levels in SVCT2(-/-), SVCT2(+/-), and SVCT2(+/+) were genotype-dependent. SVCT2(-/-) fetuses had significantly lower vitamin C levels than littermates in placenta, cortex, and lung, but not in liver (the site of vitamin C synthesis). Low vitamin C levels in placenta and cortex were associated with elevations in several markers of oxidative stress: malondialdehyde, isoketals, F(2)-isoprostanes, and F(4)-neuroprostanes. Oxidative stress was not elevated in fetal SVCT2(-/-) lung tissue despite low vitamin C levels. In addition to the expected severe hemorrhage in cortex, we also found hemorrhage in the brain stem, which was accompanied by cell loss. We found evidence of increased apoptosis in SVCT2(-/-) mice and disruption of the basement membrane in fetal brain. Together these data show that SVCT2 is critical for maintaining vitamin C levels in fetal and placental tissues and that the lack of SVCT2, and the resulting low vitamin C levels, results in fetal death and, in SVCT2(-/-) mice that survive the gestation period, in oxidative stress and cell death.

Isoform-specific effects of apolipoprotein E on atherogenesis: gene transduction studies in mice.

BACKGROUND: We recently used a bone marrow-based gene therapy approach to show that small amounts of retrovirus-derived human apolipoprotein E3 (apoE3) produced by macrophages are protective against early atherosclerosis in apoE-deficient mice. METHODS AND RESULTS: In the present study, we evaluated whether the effect produced by macrophage-derived apoE3 is related to its ability to bind cellular membranes. To this end, we used apoE2 and apoEcys142, dysfunctional human variants with reduced binding to the LDL receptor or to heparan sulfate proteoglycans, respectively. ApoE-deficient mice, 5 weeks of age, received transplants of apoE(-/-) bone marrow cells transduced with either parental retrovirus or apoE3, apoE2, or apoEcys142 retroviral vectors. Human apoE was detected by ELISA in the serum of apoE3, apoE2, and apoEcys142 mice as early as 4 weeks after bone marrow transplantation, and at 8 weeks, plasma apoE levels were 55.5+/-20.3, 50.5+/-8.7, and 15.3+/-7.3 microgram/dL, respectively. In all groups, cholesterol levels increased with age but were not affected by apoE expression. As previously demonstrated, the lesion area in male apoE3 mice (3808+/-2224 micrometer(2)/section) was 40% smaller than that in control mice (6503+/-3475 micrometer(2)/section). In apoE2 mice, however, the lesion area was similar to that of controls (5991+/-2771 micrometer(2)/section), and apoEcys142 mice showed an unexpected and significant increase in lesion size (10 320+/-6128 micrometer(2)/section). Thus, transplantation with marrow transfected with receptor binding-defective apoE variants did not replicate the antiatherogenic effect of apoE3. CONCLUSIONS: These data provide in vivo evidence suggesting that macrophage-derived apoE delays development of atherosclerosis through a receptor-dependent pathway.

Retrovirus-mediated expression of apolipoprotein A-I in the macrophage protects against atherosclerosis in vivo.

We have previously reported that the lack of apolipoprotein (apo) E expression by macrophages promotes foam cell formation in vivo. Because transgenic mice overexpressing human apoA-I from the liver (h-apoA-I TgN) are protected from the atherogenesis induced by apoE deficiency, we hypothesized that the presence of apoA-I in the vessel wall could reduce the negative effect of apoE deficiency on lesion growth. To address this issue, we used both retroviral transduction and transgenic approaches to produce in vivo systems where apoA-I is expressed from macrophages. In the retroviral transduction study, apoA-I-deficient (apoA-I(-/-)) mice reconstituted with apoE-deficient (apoE(-/-)) bone marrow cells that were infected with a retroviral vector expressing human apoA-I (MFG-HAI) had 95% lower atherosclerotic lesion area than that of recipients of apoE(-/-) bone marrow cells infected with the parental virus (MFG). To determine whether the protective effect of locally produced apoA-I was due to the lack of systemic apoA-I, we conducted a different experiment using h-apoA-I TgN mice as recipients of apoE(-/-) bone marrow with or without human apoA-I (driven by a macrophage-specific transgene defined as mphi-AI). Aortic lesion area in apoE(-/-)/mphi-AI --> h-apoA-I TgN mice was decreased by 85% compared with apoE(-/-) --> h-apoA-I TgN mice. These data demonstrate that expression of apoA-I from macrophages protects against atherogenesis without affecting plasma apoA-I and high density lipoprotein cholesterol levels.

Reduced atherosclerotic lesions in mice deficient for total or macrophage-specific expression of scavenger receptor-A.

The absence of the scavenger receptor A (SR-A)-I/II has produced variable effects on atherosclerosis in different murine models. Therefore, we examined whether SR-AI/II deficiency affected atherogenesis in C57BL/6 mice, an inbred strain known to be susceptible to diet-induced atherosclerotic lesion formation, and whether the deletion of macrophage SR-AI/II expression would modulate lesion growth in C57BL/6 mice and LDL receptor (LDLR)(-/-) mice. SR-AI/II-deficient (SR-AI/II(-/-)) female and male mice on the C57BL/6 background were challenged with a butterfat diet for 30 weeks. No differences were detected in plasma lipids between SR-AI/II(-/-) and SR-AI/II(+/+) mice, whereas both female and male SR-AI/II(-/-) mice had a tremendous reduction (81% to 86%) in lesion area of the proximal aorta compared with SR-AI/II(+/+) mice. Next, to analyze the effect of macrophage-specific SR-AI/II deficiency in atherogenesis, female C57BL/6 mice were lethally irradiated, transplanted with SR-AI/II(-/-) or SR-AI/II(+/+) fetal liver cells, and challenged with the butterfat diet for 16 weeks. In a separate experiment, male LDLR(-/-) mice were reconstituted with SR-AI/II(-/-) or SR-AI/II(+/+) fetal liver cells and challenged with a Western diet for 10 weeks. No significant differences in plasma lipids and lipoprotein profiles were noted between the control and experimental groups in either experiment. SR-AI/II(-/-)-->C57BL/6 mice, however, had a 60% reduction in lesion area of the proximal aorta compared with SR-AI/II(+/+)-->C57BL/6 mice. A similar level of reduction (60%) in lesion area was noted in the proximal aorta and the entire aorta en face of SR-AI/II(-/-)-->LDLR(-/-) mice compared with SR-AI/II(+/+)-->LDLR(-/-) mice. These results demonstrate in vivo that SR-AI/II expression has no impact on plasma lipid levels and that macrophage SR-AI/II contributes significantly to atherosclerotic lesion formation.

Macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in low density lipoprotein receptor-deficient mice.

The role of macrophage lipoprotein lipase (LPL) expression in atherosclerotic lesion formation was examined in low density lipoprotein receptor (LDLR(-/-)) mice using dietary conditions designed to induce either fatty streak lesions or complex atherosclerotic lesions. First, LDLR(-/-) mice chimeric for macrophage LPL expression were created by transplantation of lethally irradiated female LDLR(-/-) mice with LPL(-/-) (n = 12) or LPL(+/+) (n = 14) fetal liver cells as a source of hematopoietic cells. To induce fatty streak lesions, these mice were fed a Western diet for 8 weeks, resulting in severe hypercholesterolemia. There were no differences in plasma post-heparin LPL activity, serum lipid levels, or lipoprotein distribution between these two groups. The mean lesion area in the proximal aorta in LPL(-/-) --> LDLR(-/-) mice was significantly reduced by 33% compared with LPL(+/+) --> LDLR(-/-) mice, and a similar reduction (38%) in lesion area was found by en face analysis of the aortae. To induce complex atherosclerotic lesions, female LDLR(-/-) mice were lethally irradiated, transplanted with LPL(-/-) (n = 14), LPL(+/-) (n = 13), or LPL(+/+) (n = 14) fetal liver cells, and fed the Western diet for 19 weeks. Serum cholesterol and triglyceride levels did not differ between the three groups. After 19 weeks of diet, the lesions in the proximal aorta were complex with relatively few macrophages expressing LPL protein and mRNA in LPL(+/+) --> LDLR(-/-) mice. Analysis of cross-sections of the proximal aorta demonstrated no differences in the extent of lesion area between the groups, whereas en face analysis of the aortae revealed a dose-dependent effect of macrophage LPL on mean aortic lesion area in LPL(-/-) --> LDLR(-/-), LPL(-/+) --> LDLR(-/-), and LPL(+/+) --> LDLR(-/-) mice (1.8 +/- 0. 2%, 3.5 +/- 0.5% and 5.9 +/- 0.8%, respectively). Taken together, these data indicate that macrophage LPL expression in the artery wall promotes atherogenesis during foam cell lesion formation, but this impact may be limited to macrophage-rich lesions.

A direct role for the macrophage low density lipoprotein receptor in atherosclerotic lesion formation.

To evaluate the contribution of the macrophage low density lipoprotein receptor (LDLR) to atherosclerotic lesion formation, we performed bone marrow transplantation studies in different mouse strains. First, LDLR(-/-) mice were transplanted with either LDLR(+/+) marrow or LDLR(-/-) marrow and were challenged with an atherogenic Western type diet. The diet caused severe hypercholesterolemia of a similar degree in the two groups, and no differences in the aortic lesion area were detected. Thus, macrophage LDLR expression does not influence foam cell lesion formation in the setting of extreme LDL accumulation. To determine whether macrophage LDLR expression affects foam cell formation under conditions of moderate, non-LDL hyperlipidemia, we transplanted C57BL/6 mice with either LDLR(-/-) marrow (experimental group) or LDLR(+/+) marrow (controls). Cholesterol levels were not significantly different between the two groups at baseline or after 6 weeks on a butterfat diet, but were 40% higher in the experimental mice after 13 weeks, mostly due to accumulation of beta-very low density lipoprotein (beta-VLDL). Despite the increase in cholesterol levels, mice receiving LDLR(-/-) marrow developed 63% smaller lesions than controls, demonstrating that macrophage LDLR affects the rate of foam cell formation when the atherogenic stimulus is beta-VLDL. We conclude that the macrophage LDLR is responsible for a significant portion of lipid accumulation in foam cells under conditions of dietary stress.

Macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in vivo.

Expression of lipoprotein lipase (LPL) by the macrophage has been proposed to promote foam cell formation and atherosclerosis, primarily on the basis of in vitro studies. LPL-deficient mice might provide a model for testing the role of LPL secretion by the macrophage in an in vivo system. Unfortunately, homozygous deficiency of LPL in the mouse is lethal shortly after birth. Because the fetal liver is the major site of hematopoiesis in the developing fetus, transplantation of C57BL/6 mice with LPL-/- fetal liver cells (FLCs) was used to investigate the physiologic role of macrophage LPL expression in vivo. Thirty-four female C57BL/6 mice were lethally irradiated and reconstituted with FLCs from day 14 LPL+/+, LPL+/-, and LPL-/- donors. No significant differences were detected in plasma levels of post-heparin LPL activity or in serum cholesterol or triglyceride levels between the 3 groups on either a chow diet or an atherogenic diet. After 19 weeks on the atherogenic diet, aortae were collected for quantitative analysis of the extent of aortic atherosclerosis. LPL expression was detected by immunocytochemistry and in situ hybridization in macrophages of aortic atherosclerotic lesions of LPL+/+-->C57BL/6 and LPL+/--->C57BL/6 mice, but not in LPL-/--->C57BL/6 mice, whereas myocardial cells expressed LPL in all groups. The mean aortic lesion area was reduced by 55% in LPL-/--->C57BL/6 mice compared with LPL+/+-->C57BL/6 mice and by 45% compared with LPL+/--->C57BL/6 mice, respectively. These data demonstrate in vivo that LPL expression by macrophages in the artery wall promotes foam cell formation and atherosclerosis. off

Retroviral gene therapy in ApoE-deficient mice: ApoE expression in the artery wall reduces early foam cell lesion formation.

BACKGROUND: Apolipoprotein E (apoE) has long been known to play an important role in the clearance of plasma lipoproteins. More recently, a direct role for apoE in delaying atherogenesis has been proposed. Macrophage production of apoE in the artery wall has been demonstrated to provide protection against atherosclerotic lesion development independently from its role in lipoprotein clearance. However, whether macrophage apoE can affect lesion growth at all stages of atherogenesis remains to be established. METHODS AND RESULTS: To evaluate the role of macrophage apoE in different stages of atherogenesis, as well as to establish a novel gene therapy approach to atherosclerotic vascular disease, we used an apoE-expressing retrovirus to transduce apoE-deficient (-/-) bone marrow for transplantation into apoE(-/-) recipient mice. Three weeks after bone marrow transplantation, apoE was expressed from arterial macrophages and was detectable in plasma associated with lipoproteins at 0.5% to 1% of normal levels but did not affect plasma cholesterol levels. We used 2 groups of recipient mice: younger mice with lesions consisting primarily of foam cells and older mice with more advanced lesions. When either the mouse or human apoE transgenes were expressed in mice from 5 to 13 weeks of age, there was a significant reduction in lesion area, whereas no effects were detected in mice that expressed apoE from 10 to 26 weeks of age. CONCLUSIONS: We demonstrate that arterial macrophage apoE secretion can delay atherogenesis if expressed during foam cell formation but is not beneficial during the later stages of atherogenesis. These data also provide evidence that apoE transgene expression from arterial macrophages may have therapeutic applications.

Ultrastructural identification of cells with dendritic cell appearance in atherosclerotic aorta of apolipoprotein E deficient mice.

The present electron microscopic study was undertaken to see whether cells with a dendritic cell appearance accumulate in atherosclerotic lesions of apolipoprotein E (apoE) deficient mice. Atherosclerotic aortas from 7 eight-month old apoE deficient mice were examined. In atherosclerotic plaques as well as in the underlying media and adventitia, cells with a dendritic cell appearance including the presence of a unique tubulovesicular system were detected. The tubulovesicular system was most hypertrophied in their cellular processes where the continuous cisterns sometimes formed circular structures. The cells containing the tubulovesicular system lacked lysosomes and phagolysosomes and their cytoplasm was free of lipid inclusions. The present observations suggest that dendritic cells are involved in apoE deficient mouse atherosclerosis. ApoE deficient mice might be a useful model for investigating functions of dendritic cells in atherogenesis.

Hepatic apo E expression is required for remnant lipoprotein clearance in the absence of the low density lipoprotein receptor.

According to the secretion-capture model of remnant lipoprotein clearance, apo E secreted by hepatocytes into the space of Disse serves to enrich the remnants with a ligand for receptor-mediated lipoprotein endocytosis. Current evidence supports a two-receptor model of lipoprotein removal, in which apo E-containing remnants bind either the low density lipoprotein receptor (LDLR) or the LDLR-related protein (LRP). Recently, we demonstrated that reconstitution of apo E(-/-) mice with apo E(+/+) marrow results in normalization of plasma lipoprotein levels, indicating that hepatic expression of apo E is not required for remnant clearance and calling into question the relevance of the secretion-capture mechanism. To dissect the relative contributions of LDLR and LRP to the cellular catabolism of remnant lipoproteins by the hepatocyte, bone marrow transplantation (BMT) was used to reconstitute macrophage expression of apo E in mice that were null for expression of both apo E and the LDLR. Reconstitution of macrophage apo E in apo E(-/-)/LDLR(-/-) mice had no effect on serum lipid and lipoprotein concentrations, although it produced plasma apo E levels up to 16-fold higher than in C57BL/6 controls. Immunocytochemistry of hepatic sections revealed abundant staining for apo E in the space of Disse, but no evidence of receptor-mediated endocytosis of remnant lipoproteins. Transient expression of human LDLR in the livers of apo E(+/+)--> apo E(-/-)/LDLR(-/-) mice by adenoviral gene transfer resulted in normalization of serum lipid levels and in the clearance of apo E-containing lipoproteins from the space of Disse. We conclude that whereas the LDLR efficiently clears remnant lipoproteins irrespective of the site of origin of apo E, endocytosis by the chylomicron remnant receptor (LRP) is absolutely dependent on hepatic expression of apo E. These data demonstrate in vivo the physiologic relevance of the apo E secretion-capture mechanism in the liver.

Increased atherosclerosis in mice reconstituted with apolipoprotein E null macrophages.

Macrophage-derived foam cells express apolipoprotein E (apoE) abundantly in atherosclerotic lesions. To examine the physiologic role of apoE secretion by the macrophage in atherogenesis, bone marrow transplantation was used to reconstitute C57BL/6 mice with macrophages that were either null or wild type for the apoE gene. After 13 weeks on an atherogenic diet, C57BL/6 mice reconstituted with apoE null marrow developed 10-fold more atherosclerosis than controls in the absence of significant differences in serum cholesterol levels or lipoprotein profiles. ApoE expression was absent in the macrophage-derived foam cells of C57BL/6 mice reconstituted with apoE null marrow. Thus, lack of apoE expression by the macrophage promotes foam cell formation. These data support a protective role for apoE expression by the macrophage in early atherogenesis.

Cell death in atheromatous plaque of the carotid artery occurs through necrosis rather than apoptosis.

Thrombogenic plaques associated with acute myocardial infarction and sudden cardiac death are relatively acellular but the mechanisms responsible for cell loss are uncertain. To help elucidate this, we investigated advanced atherosclerotic lesions taken from 13 carotid arteries. The number of cells detected by the technique for identifying DNA fragmentation (occurring at apoptosis and necrosis) known as terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) technique varied in arteries from 9.6% to 40.3% and was 4 times higher in plaque (26.4 +/- 2.9%) than in the adjacent media (6.0 +/- 1.3%). The majority of TUNEL+ cells (75.5 +/- 5.6%) were smooth muscle cells (SMCs) as they also reacted with antibodies to SMC alpha-actin and myosin. TUNEL+ nuclei were found in macrophages (CD68+ cells), T-cells (CD3+ cells) and neovascular endothelial cells (von Willebrand factor: vWf+ cells) as well. From 13.0% to 34.0% (24.6 +/- 1.7%) of plaque cells stained with antibody to proliferating cell nuclear antigen (PCNA) whereas the number of PNCA+ nuclei in the adjacent media was 6 times lower (4.1 +/- 0.7%). In plaques, the number of PCNA+ cells correlated with the number of TUNEL+ cells (r = .66; p < .001) but the proportion of PCNA+ SMCs was relatively low (39.1 +/- 5.2%). Electronmicroscopic examination showed the presence of cells with condensed chromatin and convoluted cell surfaces in all arteries tested and the numbers of cells exhibiting these destructive alterations varied from 15.2% to 35.5% but no cells with typical expression of apoptotic death were detected. The results obtained indicate that all vascular cell types undergo cell death, with some prevalence in locations close to the plaque core and that intimal cells die through necrosis rather than apoptosis. We speculate that the imbalance in SMC death and proliferation may lead to acellular plaque formation.

Immunohistochemical study of primary and recurrent basal cell and metatypical carcinomas of the skin.

We investigated cell proliferation and expression of cytoskeletal proteins in 32 cases of primary basal cell carcinomas (BCC), 10 cases of recurrent BCC, and 10 cases of metatypical carcinomas (MTC). Paraffin-embedded biopsies were evaluated immunohistochemically with a battery of antibodies. Antibodies to proliferating cell nuclear antigen (PCNA) demonstrated comparatively low numbers of proliferating cells in 25 of 32 cases of primary BCC. In contrast, both recurrent BCC and MTC exhibited three to four times higher levels of proliferating cells than primary BCC. PCNA-positive cells were usually distributed uniformly throughout the lobules; at times, however, they were localized to the outer areas of those neoplasms, with a comparatively low level of proliferation index. Antibodies to keratin 17 strongly stained cells of all BCC cases, and antibodies to keratin 8 reacted with most of them. In contrast, the staining intensity of both types of keratin in MTC was decreased six to eight times as compared with all BCC. In addition, cells of eight BCC and three MTC reacted with antibodies to smooth muscle alpha-actin and myosin, neoplasms that did not differ by the number of PCNA-positive nuclei from carcinomas without contractile proteins. The differences in cell proliferation and keratin expression between BCC and MTC may be useful criteria for further distinguishing these carcinomas. The appearance of contractile proteins in some BCC and MTC may be the result of, or implies, myoepithelial differentiation.

[Expression of gene K51 in normal epidermis and various epithelial skin tumors in man]. / Ekspressiia gena K51 v normal'nom épidermise i nekotorykh épitelial'nykh opukholiakh kozhi cheloveka.

The expression of gene K51 in the cells of human normal epidermis and epithelial skin tumors was investigated using in situ hybridization method with radioactive probe. The K51 gene transcripts were detected in the epidermis, sebaceous and sweat glands of human embryo and adult skin. The level of gene expression was higher in the stratum granulosum than in the basal layer of the skin. K51 gene expression was also found in the basal cell and metatypical carcinomas, with the level of expression lower than in the neighbouring epidermis and higher than in the surrounding skin stromal cells. Thus, K51 gene is expressed in the skin epidermis of human embryo and adults but the level of its activity is dramatically decreased in the cells of skin epithelial tumors. This potentially may be important as a diagnostic test.

[Proliferative activity of basal cell and metatypical cell carcinoma of the skin]. / Proliferativnaia aktivnost' bazal'nokletochnogo i metatipicheskogo raka kozhi.

Using avidin-biotin, immunoperoxidase techniques and antibodies to proliferating cell nuclear antigen (PC10), 32 cases of primary basal cell carcinoma (BCC), 10 cases of recurrent BCC and 8 cases of metatypical carcinoma (MTC) of the skin were studied. The majority of cases of primary BCC had low level of cell proliferative activity, whereas high cell proliferative index was found only in 7 cases. Recurrent BCC as well as MTC exhibited high level of cell proliferative index that 3-4 times exceeded the same index of primary BCC. BCC had either uniform or regional distribution of proliferative cells. Tumors with regional distribution of proliferative cells are characterized by a low level of proliferation. The revealed difference in cell proliferation between primary BCC, recurrent BCC and MTC may be valuable criterion for detecting the degree of tumor malignancy.

Monocyte/macrophage accumulation and smooth muscle cell phenotypes in early atherosclerotic lesions of human aorta.

In a search for early atherosclerotic lesions, we have investigated grossly normal areas of human thoracic aortas taken at autopsy from 40 trauma victims aged from 3 to 40 years. Two areas of aorta were compared: lesion predisposed to atherosclerosis (LP) area localized on the dorsal aspect of the vessel along the row of intercostal branching sites, and lesion resistant (LR) area located on the ventral aspect of the vessel. Accumulation of apolipoprotein B (apo B) was found in LP aortic area of each child older than 6 years. Similar retention of apo B in LR area appeared only in aortas of teenagers. The apo B staining increased with age in both areas tested but was usually of a greater extent in LP area than in LR area. Typical smooth muscle cells (SMCs) and a few monocytes/macrophages (Mn/Mph) were revealed in the intimal layer of all aortas examined. The number of Mn/Mph dramatically increased in LP areas of individuals over 17 years. Quantitative study of double stained sections has shown a 2- to 6-fold enhanced number of Mn/Mph in LP area compared with LR aortic area of 10 men over 21 years. Focal infiltration of Mn/Mph in aortas of young adults occurred without endothelial denudation. In addition, some intimal SMCs in LP area of 12 aortas out of 29 expressed desmin and contained well-developed endoplasmic reticulum, while such cells were seldom detected in LP area of the vessels. Thus, focal accumulation of apo B with subsequent Mn/Mph infiltration and SMC phenotypic modulation in LP aortic area of young adults may be causally involved in fatty streak and atherosclerotic plaque formation.

Phenotype related changes of intimal smooth muscle cells from human aorta in primary culture.

To study the functional characteristics of smooth muscle cell (SMC) phenotypes, we have investigated myosin expression, cell proliferation, collagen production and low-density lipoprotein (LDL) receptor activity in intimal SMCs of normal human aorta during their growth in primary culture. By staining with rabbit antibodies to smooth muscle myosin (ASMM) 3 cell types could be distinguished in culture: homogeneously stained cells, cells with discontinuous myosin fibrils and myosin-negative cells. The ratio of cell types greatly changed with culture growth: on days 5, 7 and 14 it was 82:1:17%, 70:5:25% and 10:30:60%, respectively. After 5-6 days of culture intimal SMCs began to proliferate and DNA-synthesizing nuclei were seen 1.5-4.3 times more frequently in myosin-negative cells than in cells with homogeneous myosin distribution. At that time the number of cells reacted with monoclonal antibody (MAb) to an epitope shared collagen types I and III started to increase. By double immunofluorescence staining it was shown that the cultured cells containing both ASMM and MAb markers were found 2.0-4.8 times more rarely than MAb-positive staining in myosin-negative cells. During the first 5 days in culture LDL binding and uptake were diminished in intimal cells with intercellular lipid inclusions independently of their myosin staining pattern, but their activity increased with culture growth. Thus, SMCs from human aortic intima change their phenotype on days 6 and 7 in primary culture as manifested by alteration of myosin expression, increased cell proliferation, collagen production and LDL receptor activity. Changes in myosin expression, however, are not an essential prerequisite for cell proliferation and collagen production.